First record of Fusarium concentricum (Hypocreales: Hypocreaceae) isolated from the moth Polychrosis cunninhamiacola (Lepidoptera: Tortricidae) as an entomopathogenic fungus

Abstract Fusarium concentricum Nirenberg & O’ Donnell (Ascomycota: Hypocreales) is a fungal species known to infect plants, but never reported as entomopathogenic. Polychrosis cunninhamiacola Liu et Pei (Lepidoptera: Tortricidae: Olethreutinae) is a major and widespread insect pest causing economic losses to cultivated Chinese fir Cunninghamia lanceolata (Lamb.) Hook. It is routinely controlled by extensive use of chemical insecticides, which is perceived as environmentally unsustainable. During March and April of 2019–2020, muscardine cadavers of larvae and pupae of P. cunninhamiacola infected with growing fungus were collected in a fir forest in northern Guangdong Province, China. Conidia were isolated and cultured on PDA medium, from which the fungal strain was identified as F. concentricum FCPC-L01 by morphology and by sequence alignment match with Tef-1α gene. Pathogenicity bioassays at the conidial concentration 1 × 107 revealed P. cunninhamiacola adults and Danaus chrysippus (L.) (Lepidoptera: Nymphalidae) larvae are sensitive to the fungal infection, but not the fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae). We believe results indicate this fungal strain might be applicable against specific target insect pests. As this is the first record of a natural infection caused by F. concentricum in insects, we propose host specificity tests should be done to evaluate its potential as a biocontrol agent.


Introduction
The Chinese fir Cunninghamia lanceolata (Lamb.) Hook is a tree endemic to China reputed for its pronounced tolerance to ambient poor soil stress, growth speed, and quality of wood (Rong-Li et al. 2014). It is one the fastest growing timber species across southern provinces in China (Zeng et al. 2006). Nonetheless, the overall wood quality and seed yield of C. lanceolata has been threatened due to an increase in the number of diseases and pests affecting this species (Yu 2000):to date, there are 7 different diseases and 5 species of pest insects reported in China (Tian et al. 2019). For instance, the moth Polychrosis cunninhamiacola (Lepidoptera: Tortricidae: Olethreutinae) inflicts major losses to growers of C. lanceolata in China, and this pest has spread into southern provinces of China during the latest years, reaching, for example, the city of Shaoguan in Guangdong province (Liu et al. 2007, Peng et al. 2021). This moth is widespread across the C. lanceolata-producing provinces of Guangdong, Guangxi, Fujian, Jiangxi, Zhejiang, Guizhou, and Sichuan. Females of P. cunninhamiacola lay their eggs on the leaves of C. lanceolata and the hatching larvae travel to the top buds of young shoots, causing deleterious effects and marked malformations such as multiple apical shoots at lower height and trunk bending (Liu et al. 2007), also seriously impairing growth speed and wood quality (Fig. 1). Copious amounts of chemical pesticides are routinely used in attempting to control this insect pest, however since the planted areas of C. lanceolata are so vast, the practice has become also a significant concern because of the generated pollution that can severely affect the environment, the safety of forest-derived products, and kill natural enemies such as parasitic wasps (Hamburg and Guest 1997). Therefore, novel pest control strategies are now a necessity for managing forests composed of C. lanceolata.
Entomopathogenic fungi can infect and kill destructive insects, and thus play a central role in Integrated Pest Management (IPM) by offering a more natural protection through natural enemies, leading towards an ecological balance (Gul et al. 2014, Kidanud 2020. The fungal genera Beauveria and Metarhizium are two important entomopathogenic agents employed in controlling agricultural and forestry pest insects because of their wide host ranges (Meyling and Eilenberg 2007). Also, some strains of the genus Fusarium have been reported as efficient in controlling pest insects, exhibiting some of the characteristics desirable for an agent of agricultural and forestry biological control, such as delivering high mortality rates and presenting abundant sporulation capacity. Fusarium fungal strains are badly reputed as some species are considered plant pathogens and producers of mycotoxins (Mohanty et al. 2008, Munshi et al., 2008, Pelizza et al. 2011. To date, nine species of Fusarium have been demonstrated to be pathogenic to insects, attacking hosts within Lepidoptera, Diptera, Coleoptera, Hemiptera, Hymenoptera, and Orthoptera (Santos et al. 2020).
In this study, we isolated F. concentricum from dead moths and tested its toxicity against living P. cunninhamiacola and the other pest species Danaus chrysippus L.(Lepidoptera), and Solenopsis invicta Buren (Hymenoptera). The species F. concentricum belongs to the F. fujikuroi species complex, and is the most common fungus causing the fruit blotch in the roselle Hibiscus sabdariffa, and the pepper fruit rot in Capsicum annuum (Wang et al. 2013, Rahim et al. 2020. This species has never been recorded attacking insects. This research attempted to evaluate the potential for development and utilization of F. concentricum as an alternative biological agent for pest management.

Cadaver Collection and Species Identification of Fusarium Strain FCPC-L01
During the months of March and April of 2019 and 2020, the amount of damage inflicted by pest insects to a seed garden of C. lanceolata was evaluated in the northern part of the city of Shaoguan, Guangdong province of China (coordinates N:24.705, E:113.825). A large number of dead larvae and pupae of P. cunninhamiacola showing signs of fungal infection and sprouting white hyphae were found (Fig. 2). Careful inspection of conidial morphology under the microscope indicated that the growing fungi were neither Beauveria adult， (e-f) attacked fir branch producing multiple shoots at lower height indicated with arrows. Unharmed trees usually present only one shoot at lower height for each tree; the onset of several shoots after attack by P. cunninhamiacola larvae will significantly affect tree growth.
bassiana nor Metarhizium anisopliae, based on the presence of crescent shaped macro-and micro-conidia.
The conidia on the surface of cadavers were carefully sampled with a thin wire needle and seeded into a 2 ml tube containing 0.05 % Tween-80 solution, from which 0.1 ml -aliquots were spread onto Potato-Dextrose Agar medium (PDA, 200 g/L potato, 20 g/L dextrose, and 20 g/L agar) in 9-cm wide Petri dishes using a triangular glass rod (Du et al. 2019). The inoculated Petri dishes were incubated at 25 ± 2℃, relative humidity 75 ± 5% for 3-6 days. Sprouting hyphae were collected from a single colony in the plate,  subcultured in a new PDA plate to produce a pure culture, and incubated for 8 days.
The fungi was further identified as genus Fusarium based on the elongation factor 1α (TEF-1α) (Kristernsen et al. 2005). DNA from Fusarium strain FCPC-L01 (isolated from larvae of P. cunninhamiacola) was extracted using a genomic DNA extraction kit (Axygen Biotechnology, Hangzhou, China). TEF-1α sequences of Fusarium strains were amplified by PCR using a set of primers (from Du et al. 2019): TEF-1αF:5ʹ-ATGGGTAAGGAGGACAAGAC-3ʹ and TEF-1αR:5ʹ-GGAAGTACCAGTGATCATGTT-3ʹ. The amplified product was sequenced by the Sanger method at Shenggong, Ltd, Gaugnzhou, China. The cloned TEF-1α gene sequences were aligned using ClustalW with MEGA5 software (Tamura et al. 2011). Gene homology was assessed using NCBI BLASTn queries and a phylogenetic tree constructed by Neighbor-Joining method using MEGA5 software with Fusarium FCPC-L01 and other model Fusarium strains and the outgroup M. anisopliae. The sequence was submitted to the GenBank and accession number (Genbank accession OP617256) was obtained.

Preparation of Conidial Suspension
The Fusarium strain FCPC-L01 was maintained in Petri dishes containing PDA. Dishes were incubated at the constant temperature of 25 ± 1°C, 85 ± 1% RH for 8 days in an incubator. Conidia were carefully sampled with a sterilized brush and suspended in sterilized 0.05% Tween-80 water solution. The conidial concentration was measured using a haemocytometer, and adjusted to 1 × 10 7 conidia/ ml for further experiments.

Pathogenicity Bioassay
Adults of P. cunninhamiacola were obtained from the C. lanceolata seed garden of Shaoguan city, larvae of D. chrysippus were also collected locally, nearby a seed garden, and workers of the fire ant Solenopsis invicta were collected from a public park in Guangzhou, China. All sampled insects were contained in plastic boxes (50 cm × 40 cm × 15 cm) placed in the dark in a thermal incubator set to 25 ± 1°C and 85 ± 1%relative humidity (RH). The fire ants were fed ad libitum with Tenebrio molitor larvae and 25% sucrose water, the lepidopteran larvae were fed leaves of their host plants and the adults fed 25% honey every other day.
For bioassays, samples of the different insects were placed in micro-centrifuge tubes containing conidial suspensions at the set concentration of 1 × 10 7 conidia/ml. Specifically, for each species, a control group (N = 60) was treated with 0.05% Tween-80 aqueous solution. Per experimental groups, insects (N = 60) of each species were submerged into the conidial solution and gently swirled for five seconds, after which the excess liquid was removed from their surface by dabbing with a piece of filter paper for 10 min. The insects were then introduced into separated plastic boxes and kept in groups based on their treatments, and maintained under light: dark = 12h: 12h conditions at 25 ± 1°C and 85 ± 1% RH for 8 days. The mortality within each experimental group was recorded daily and the dead insects were removed to reduce the possibility of cross-contamination by Fusarium conidia. Removed cadavers were surfacesterilized and monitored for sprouting conidia for another 8 days.

Plant Infection Bioassays
The Fusarium strains FCPC-L01, SM-LK-2, and SM-LK-b5 were selected for plant infection bioassays. SM-LK-2, and SM-LK-b5 were isolated from plants of C. lanceolata. Aqueous solution of 0.05% Tween-80 was used as control. Preparation of conidial suspensions of SM-LK-2 and SM-LK-b5a was the same as described above of FCPC-L01 for the treatment of the experimental insects. Leaves of healthy C. lanceolata seedlings (10-cm high) were sprayed with 2-mL conidial suspensions (10 7 spore/ ml) (N = 10) of one the different strains. The control was 0.05% Tween-80 aqueous (N = 10). The plants were then maintained under 12: 12 h dark: light conditions at 25 ± 1°C and 75 ± 1% RH. The morphological state of the plants such as the color of leaves and mycelium growth on the surface of leaves in each group were recorded daily for 7 days.

Statistical Analysis
All collected data were analyzed using SPSS v.22.0. Mortality bioassays based on the median lethal time (LT 50 ) were examined using Probit regression analysis. The survivorship distributions at different treatment groups were compared by the Breslow statistics (Kaplan-Meier survival test), and the hazard ratio of death was analyzed using the Cox Proportional Regression analysis to generate the Wald Statistic.
Overall, 85% of the fungus-exposed dead lepidopterans developed F. concentricum hyphae (Fig. S3 in Supplementary files), but none within control groups, indicating fungal infection was likely the cause of death.

Plant Infection Bioassay
The effects from exposure to Fusarium conidia on C. lanceolata are shown in Fig. 6. On the 7th day posttreatment with conidial suspensions of either FCPC-L01 or Tween-80 control, no disease symptoms were observed (Fig. 6a) nor mycelia were observed on leaves (Fig. 6b). On the other hand, SM-LK-b5a seedlings showed evident wilting and hyphae forming on the leaves surface (Fig. 6b).
Regarding SM-LK-2, no mycelia were noted on leaf surfaces, while leaf chlorosis was evident.

Discussion
Entomopathogenic fungi are important biological control agents for use in agricultural and forestry pest management. In this study, dead P. cunninhamiacola larvae and pupae infected with an entomopathogenic fungus in the field were collected, which was isolated and identified as F. concentricum, morphologically and molecularly. The obtained F. concentricum strain proved pathogenic against adults of P. cunninhamiacola and larvae of D. chrysippus, but showed low toxicity against S. invicta workers in the laboratory.
As far as we know, this is the first record of natural infection by F. concentricum in insects. Correct species identification of Fusarium samples can be challenging based on morphology alone (Santos et al. 2020), as the approach grossly underestimates the extreme diversity of species in this genus. Therefore, molecular phylogeny is advisable to enhance accurate identification of Fusarium species (Chandra et al. 2011). The choice for rDNA internal transcribed spacer (ITS) sequence polymorphisms is often used for fungus identification, particularly in the Metarhizium genus (Nishi et al. 2011), however, as there is considerably less ITS polymorphism in Fusarium (Chandra et al. 2011) the gene TEF-1α was chosen because it contains enough polymorphism for species identification in Fusarium (Divakara et al., 2014).
Previous proposals for using Fusarium spp. as biological control agents have been largely dismissed, given the fact that numerous species produce harmful mycotoxins such as deoxynivalenol, trichothecenes, moniliformin, and zearalenone, potentially harmful to humans and the environment (Mello et al. 1999). Notwithstanding, it is also a fact that the technical understanding of the mycotoxins produced by commercially-available fungal biological control agents is scant, which currently include mycoinsecticides, mycoherbicides, and mycoparasites. In addition, a large body of knowledge regarding mycotoxins produced by Fusarium now exists, which should facilitate the selection of benign Fusarium strains by surveying the presence of mycotoxins through high sensitivity detection methods such as GC-MS and LC-MS (Stpień et al. 2019).
In order to sustain responsible use of insect-pathogenic fungi like Fusarium as biological control agents, it is also important to keep track of both target and non-target organisms being affected by each application system and context. For example, Fusarium oxysporum isolated from the brown planthopper Nilaparvata lugens showed no infection capacity against the host plants rice, cotton, or tomato, which is an indication that F. oxysporum strain should be safe for the host plants but infective against N. lugens (Kolander 2010). Similarly, the use of a saprophyte Fusarium species resulted in a reduction in the numbers of eggs, larvae, and adults of the pest bug Daktulosphaira vitifoliae (Hemiptera: Phylloxeridae) on grapes, but did not produce any infections on the host plant (Morquer and Nysterakis 1944). However, some strains of F. oxysporum isolated from Sitona hispidula (Coleoptera: Curculionidae) proved not only pathogenic to the insects' larvae, but also to the ornamental clover Trifolium pretense (Kilpatrick 1961). In the present study, FCPC-L01 strain delivered high mortalities to two species of lepidopteran pests. However, the strain did not infect any branches or leaves of the host plant fir C. lanceolata. FCPC-L01 infection resulted in low susceptibility against S. invicta, which suggest FCPC-L01 might pose low risk of infection against ants in forested areas. It should be emphasized that such susceptibility tests were conducted under optimal conditions for fungal growth. Further bioassays are needed against a wider range of insect species which are known to exist in the fir field where this strain was obtained, in order to provide a more realistic preview of expectable outcomes on non-target insects.
In conclusion, this study was the first to record F. concentricum attacking P. cunninhamiacola moths, extending the body of knowledge about the pathogenic capacity of this Fusarium species against lepidopterans. Future studies could evaluate mycotoxin production against nontarget insects and associated potential effects on plants and humans, as to expand our understanding of the potential to exploit host-Fusarium-environment interactions.